Obesity is a world-wide health problem that is associated with metabolic syndrome, including insulin resistance and the development of non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by hepatic steatosis and varying degrees of inflammation and fibrosis. Increasing evidence suggests that saturated free fatty acids (FFA), by promoting insulin resistance and mediating the progression from simple steatosis to steatohepatitis, are a causative link between obesity and NAFLD. However, the molecular mechanisms responsible for the effects of saturated FFA are incompletely understood. Our long-term goal is to understand the molecular mechanisms by which saturated FFA contribute to the development of NAFLD. Saturated FFA are a major source of metabolic stress that induce activation of the c-Jun NH2-terminal kinase (JNK) pathway, and gene deletion and pharmacological inhibition have established an important role for the JNK signaling pathway in obesity-induced insulin resistance and the pathogenesis of NAFLD. Our preliminary studies identified the MAP3K mixed-lineage kinase 3 (MLK3) as an important mediator of saturated FFA- induced JNK signaling. Thus, we have demonstrated that MLK3 mediates FFA-induced JNK activation, promotes obesity-induced insulin resistance and modulates FFA-induced ER stress response in specific tissues. These data indicate that MLK3 plays a critical role in the development and progression of NAFLD, most likely through metabolic stress signaling. The central hypothesis is that MLK3 contributes to the pathogenesis of NAFLD by mediating saturated FFA-induced JNK activation, thereby inducing inflammatory gene expression, promoting insulin resistance, and modulating ER stress-induced cell death in specific tissues. This hypothesis will be tested by three complementary Aims. In Aim 1 biochemical and molecular biological methods will be used to delineate the pathway(s) required for MLK3 activation by FFA. In Aim 2 biochemical and molecular biological methods will be used to determine the role of MLK3 in the FFA-induced ER stress response. In Aim 3 reciprocal bone marrow transplantation studies will be used to examine the tissue specific role of MLK3 in the development of insulin resistance. Accomplishing the goals of this proposal will increase understanding of the role of MLK3 in the pathogenesis of NAFLD. This information will provide the foundation for the potential development of novel therapeutic strategies for the treatment of this pathology.